Berg Ann-Kathrin, Hahn Elisabeth M, Speichinger-Hillenberg Fiona, Grube Annemaria Silvana, Hering Nina A, Stoyanova Ani K, Beyer Katharina
Department of General and Visceral Surgery, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, 10117 Berlin, Germany.
Cancers (Basel). 2023 Mar 15;15(6):1773. doi: 10.3390/cancers15061773.
Despite intensive scientific efforts, the therapy of peritonitis is presently limited to symptomatic measures, including infectious source control and broad-spectrum antibiotics. Promising therapeutic approaches to reduce morbidity and mortality are still missing. Within the early phase of abdominal sepsis, apoptosis of neutrophil granulocytes is inhibited, which is linked to tissue damage and septic shock. TNF-related apoptosis-inducing ligand (TRAIL) is a promising agent to stimulate neutrophil apoptosis. However, the underlying mechanisms have not been elucidated so far. The objective of the present study was to characterize the molecular mechanisms of TRAIL-stimulated apoptosis in early abdominal sepsis. Therefore, the murine sepsis model Colon ascendens stent peritonitis (CASP) was applied in wild type (WT) and TRAIL knock-out (TRAIL-/-) C57/BL6j mice. Neutrophil granulocytes were isolated from spleen, blood, bone marrow, and peritoneal lavage using magnetic-activated cell sorting. Neutrophil maturation was analyzed by light microscopy, and apoptotic neutrophils were quantified by fluorescence-activated cell sorting (FACS). Western blot and FACS were used to investigate expression changes in apoptotic proteins and TRAIL receptors. The impact of TRAIL-induced apoptosis was studied in vitro. In septic mice (CASP 6 h), the number of neutrophils in the BM was reduced but increased in the blood and peritoneal lavage. This was paralleled by an increased maturation of neutrophils from rod-shaped to segmented neutrophils (right shift). In vitro, extrinsic TRAIL stimulation did not alter the apoptosis level of naïve neutrophils but stimulated apoptosis in neutrophils derived from septic WT and TRAIL-/- mice. Neutrophils of the bone marrow and spleen showed enhanced protein expression of anti-apoptotic Flip, c-IAP1, and McL-1 and reduced expression levels of pro-apoptotic Bax in neutrophils, which might correlate with apoptosis inhibition in these cells. CASP increased the expression of intrinsic TRAIL in neutrophils derived from the bone marrow and spleen. This might be explained by an increased expression of the TRAIL receptors DR5, DcR1, and DcR2 on neutrophils in sepsis. No differences were observed between septic or naïve WT and TRAIL-/- mice. In conclusion, the present study shows that neutrophil granulocytes are sensitive to TRAIL-stimulated apoptosis in the early stage of abdominal sepsis, emphasizing the promising role of TRAIL as a therapeutic agent.
尽管进行了大量科学研究，但目前腹膜炎的治疗仍局限于对症治疗措施，包括控制感染源和使用广谱抗生素。降低发病率和死亡率的有效治疗方法仍然缺乏。在腹部脓毒症的早期阶段，中性粒细胞的凋亡受到抑制，这与组织损伤和感染性休克有关。肿瘤坏死因子相关凋亡诱导配体（TRAIL）是一种有潜力刺激中性粒细胞凋亡的药物。然而，其潜在机制迄今尚未阐明。本研究的目的是明确TRAIL在早期腹部脓毒症中刺激凋亡的分子机制。因此，在野生型（WT）和TRAIL基因敲除（TRAIL-/-）的C57/BL6j小鼠中应用了小鼠脓毒症模型升结肠支架性腹膜炎（CASP）。使用磁珠分选法从脾脏、血液、骨髓和腹腔灌洗液中分离中性粒细胞。通过光学显微镜分析中性粒细胞的成熟情况，并用荧光激活细胞分选法（FACS）对凋亡中性粒细胞进行定量。采用蛋白质免疫印迹法和FACS研究凋亡蛋白和TRAIL受体的表达变化。在体外研究了TRAIL诱导凋亡的影响。在脓毒症小鼠（CASP 6小时）中，骨髓中的中性粒细胞数量减少，但血液和腹腔灌洗液中的中性粒细胞数量增加。与此同时，中性粒细胞从杆状向分叶状中性粒细胞的成熟增加（右移）。在体外，外源性TRAIL刺激并未改变未成熟中性粒细胞的凋亡水平，但刺激了脓毒症WT和TRAIL-/-小鼠来源的中性粒细胞的凋亡。骨髓和脾脏中的中性粒细胞显示抗凋亡蛋白Flip、c-IAP1和McL-1的蛋白表达增强，而促凋亡蛋白Bax在中性粒细胞中的表达水平降低，这可能与这些细胞中的凋亡抑制有关。CASP增加了骨髓和脾脏来源的中性粒细胞中内源性TRAIL的表达。这可能是由于脓毒症中中性粒细胞上TRAIL受体DR5、DcR1和DcR2的表达增加所致。在脓毒症或未感染的WT和TRAIL-/-小鼠之间未观察到差异。总之，本研究表明，在腹部脓毒症早期，中性粒细胞对TRAIL刺激的凋亡敏感，强调了TRAIL作为治疗药物的潜在作用。
